PROJECT SUMMARY Prostate and pancreatic cancers are among the most common and difficult to treat cancers. Major dilemmas in the management of prostate cancer include the difficulty of discriminating between aggressive and indolent forms of the disease and the need for improved treatment of high-risk and castrate resistant metastatic disease. In the case of pancreatic cancer, the major problems are late diagnosis and high lethality. One major barrier to progress and an unmet need in both diseases is the relative absence of effective molecular imaging tracers/tools that can be used at the whole-body or intraoperative level, to guide patient management. Antibodies can provide highly specific probes for molecular targets, and can be engineered to optimize their utility as imaging agents for clinical use. During the previous project period, novel immunoPET imaging agents have been developed based on engineered antibody fragments directed towards Prostate Stem Cell Antigen (PSCA), a cell surface biomarker expressed by a majority of prostate and pancreatic cancers which is also a promising therapeutic target. Two fragment formats, cys-diabody and cys-minibody, which enable site-specific conjugation and labeling, have been produced with the goal of providing multifunctional fragments that can be radiolabeled with positron-emitting radionuclides 124I, 89Zr, or 18F, and/or conjugation with fluorescent labels for optical imaging. In the proposed work, fluorescently labeled PSCA cys-diabodies and cys-minibodies will be developed as intraoperative molecular imaging probes and their utility assessed in preclinical models. Dually labeled probes (PET/optical) will also be produced such that the same probe can be used for whole-body imaging followed by intraoperative visualization of local/regional spread. Finally, based on promising biodistribution and imaging studies of minibodies in patients, the therapeutic potential of radiolabeled anti-PSCA cys-minibodies will be explored, comparing non-residualizing (131I) and residualizing (177Lu) therapeutic radionuclides. Full biodistributions will be performed for dose estimations (mouse and human dosimetry), followed by radioimmunotherapy studies in preclinical models. Furthermore, the efficiacy of PSCA-targeted radioimmunotherapy in combination with androgen ablation or PARP inhibition will be evaluated. These fully humanized immunoPET probes can be readily translated to the clinic to address pressing questions in clinical management, including staging of newly diagnosed, recurrent and metastatic prostate and pancreatic cancers; improved selection and classification of patients for PSCA-targeted therapy; and monitoring disease response to therapy, and ultimately could serve as an efficacious delivery vehicle for tumor-targeted radiotherapy.